Protein Analysis 1 Paper Chromatography and Electrophoresis
In order to analyze a protein, the protein is usually hydrolyzed, breaking the peptide linkages to release the individual amino acids
The peptide bond is rather strong. Acid hydrolysis may requires the use of 6.0 M hydrochloric acid at 1100C for 1 to 3 days.
Hydrolysis of the peptide linkages produces individual amino acids, which can be identified using electrophoresis and
chromatography. Chromatography is a very useful method for the separation of mixtures of substances, which are otherwise not
readily separated.
1. Paper Chromatography
Paper chromatography may be used to identify the components of a very small sample. It is particularly suitable for separating
hydrophilic substances such as amino acids. The water in the paper fibers acts as the stationary phase as the solvent flows by
capillary action up the paper in ascending chromatography or down the paper in descending chromatography.
Amino acids can be separated as a result of a phenomena known as adsorption. Adsorption involves the concentration of one
substance at the surface of another. In the case of paper chromatography, separation results from the partition of the amino acid
between two solvents. One solvent, water (the stationary phase) is adsorbed on the cellulose, which makes up the paper. The other
solvent travels up the paper. It is called the eluting solvent. The eluting solvent is usually a mixture of an alcohol and a polar solvent
such as 1-butanol and acetic acid or ammonia and isopropyl alcohol. Each amino acid has a particular solubility in the eluting
solvent and the stationary phase. Thus amino acids with greater solubility in the eluting solvent will travel higher up in ascending
chromatography.
Experimentally, a solution of the sample of the mixture of amino acids to be analyzed is placed as a spot on the surface of the
chromatographic paper a couple of centimeters from the bottom and marked in pencil.
The solvent is allowed to evaporate from the spot. Then the chromatography paper is placed vertically in a covered container. The
solvent travels up the paper due to capillary action. The components of the mixture move with the solvent at different rates
depending on their solubility in the stationary and moving phases. Once the solvent reaches near the top of the paper it is removed
and the location of the solvent front is recorded. The solvent on the paper is allowed to evaporate.
Since amino acids are colorless, the plate must be developed. A solution of ninhydrin is used for this purpose. Glycine, for example,
forms a blue/purple compound with ninhydrin, as do 19 of the 20 protein-derived a-amino acids (proline gives an orange color).
Once the positions of the spots are located the Rf value is determined. Rf refers to the “Ratio of fronts” It is the ratio of the distance
traveled by a compound (dc) over the distance traveled by the solvent (ds). Rf values of the spots can be compared with those for
pure amino acids developed at the same time, under the same conditions of solvent and temperature. Different substances have
different Rf values under similar experimental conditions, so comparison of Rf values allows for the components of a mixture to be
identified. When several components of a mixture have similar Rf values using a particular solvent making complete separation
impossible, two-dimensional chromatography may be used to improve the results. In this case, the sample spot is placed in one
corner of a square piece of paper. The chromatogram is developed by eluting with one solvent system to allow partial separation.
The paper is dried, turned at right angles from its original position and developed using a second solvent system (such as 2butanol/ammonia mixture) to achieve a more complete separation.
Procedures
The following substances will be available for your use
Amino Acids
Glycine
Alanine
Cysteine
Leucine
Glutamic Acid
Amino Acid mixtures
Solvents
Isopropyl
alcohol
1-butanol
acetic acid
Ammonia
Developers
2% Ninhydrin
Develop a procedure to run paper chromatograms for these solutions. You may experiment with various solvents. Which solvent
systems provide the best separation? What differences do you see in the behavior of the amino acids used?
2. Electrophoresis
Electrophoresis is a method of separating similarly sized molecules on the basis of their charge, The R sides chain of amino acids
varies. Some side chains are organic carbon chains. For example R = –CH3 in alanine. These amino acids are considered neutral. .
Other amino acids contain basic groups in the side chain. For example lysine contains a second -NH2 group. Other amino acids
such as aspartic acid contain a second carboxylic acid group –COOH. The presence of the basic and acidic side chains produces
positively and negatively charged ions respectively in the peptide linkage. How an amino acid, polypeptide or protein behaves in the
presence of an electric field depends on the relative numbers of these positive or negative functional groups. These groups are
further influenced by pH of the solution
The Isoelectric Point
The isoelectric point, or pI, of an amino acid (and a protein) is the pH at which the positive and negative charges are exactly
balanced. In this case the molecule has no net change and it shows no net migration in an electric field at that pH.
The table below lists isoelectric points, pI, of some amino acids
Amino Acid pKa
pKb
pK side pI
chain
Glycine
2.3
9.6
6.0
Alanine
2.3
9.7
6.0
Glutamine
2.2
9.1
5.7
Cysteine
1.8
10.8
8.3
5.1
Lysine
2.2
9.2
10.8
9.7
Histidine
1.8
9.2
6.0
7.6
An amino acid is least soluble at its isoelectric point. At this point the amino acid molecules, which have a net charge of zero, can
join together and precipitate. At a lower pH, the basic group(s) attract H+ to form -+NH3, i.e., a positive charge. At a higher pH, the
acidic R group on the amino acid donates its H+ to produce COO-, i.e., a negative charge. Therefore the solubility of an amino acid
increases at pH values higher or lower than its isoelectric point.
pH = 3 pH =6 pH = 10
The pI of alanine is about 6.0. The diagram above shows the forms of alanine at acidic, basic and nearly neutral pH's. At pH of 5.0
the zwitterions predominates. At a pH of 2 the positive ion predominated and at pH of 10 the negative ion predominates
Electrophoresis of Amino Acids
A mixture of amino acids with different isoelectric points can be separated using electrophoresis. Such a separation can be carried
out using paper, cellulose acetate, certain types of gels, or other appropriate solid supports. The solid support is saturated with a
buffer solution of known pH.
The sample consisting of a mixture of amino acids is applied to the center of the paper. The positive and negative electrodes and
ends of the paper are placed in the buffered solution. An electric potential is applied to the electrodes.
Any amino acid that is at its isoelectric point will not move in either direction. Amino acids that have positive charges at the pH of the
buffer will move to the cathode and amino acids that have negative charges at the buffer pH move to the anode.
At a pH of 6.0, for example, alanine exists as the zwitter ion, H3N+-CH (CH3)-COO-. If an electrophoresis is carried out with alanine
at pH = 7.0, the pH of the buffer is more basic than the isoelectric point of alanine (7.0 compared to 6.0), alanine will have a net
negative charge and it will migrate toward the positive electrode. However, if the electrophoresis is carried out at pH = 5.0, the pH of
the buffer is more acidic than the isoelectric point of alanine (5.0 compared to 6.0), alanine will have a net positive charge and
migrate to the negative pole.
Example
If electrophoresis of a mixture of the six amino acids listed in the table above is carried out at a pH of 6.0, the following would
be observed:
1. Glycine and alanine will not move from the point of origin since they have net charges of zero at their isoelectric
points of 6.0.
2. Cysteine (pI = 5.1) and glutamine (pI = 5.7) will have negative charges since the buffer pH of 6.0 is more basic and
will move to the positive pole.
3. Histidine (pI = 7.6) and lysine (pI = 9.7) will have more positive charges since the buffer of 6.0 is more acidic and will
move to the negative pole.
The greater the difference in pH, the faster the migration. When sufficient separation is achieved, the paper strip can dried and
developed with the ninhydrin solution to make the amino acid components visible.
Protein electrophoresis
Electrophoresis can also be used to separate and purify proteins based on their size, since smaller protein molecules can move
more easily through the gel than larger ones. In this technique, a protein mixture sample is treated with SDS (sodium dodecyl
sulphate), which gives the proteins a negative charge. In the presence of an electric charge, the negatively charged protein
molecules migrate to the positive anode at a rate, which depends on the size of the protein molecule. This then can be compared to
known samples to identify the protein. This method also separates the different proteins, thus purifying them.
Procedures
The following substances will be available for your use
Amino Acids
Buffers
Developers
Glycine
Alanine
Cysteine
Leucine
Glutamic Acid
Amino Acid mixtures
pH = 10
pH = 7
pH =4
2% Ninhydrin
Some 9-volt batteries with alligator clips and possibly a DC power supply will be available. Devise a procedure to run electrophoresis
solutions of the above amino acids. You will need to devise your own procedure, choose the appropriate materials and plan for
proper controls. Observe the behavior of the amino acids with the three different pH levels. Compare your results to those you
obtained for chromatography